Multifluorescence confocal imaging typically utilizes a multi-channel microarray scanner to obtain images of dye spots of a microarray. As illustrated in FIG. 1, microarrays are created with fluorescently labeled DNA samples in a grid pattern consisting of rows 22 and columns 20 typically spread across a 1 by 3 inch glass microscope slide 24. Each spot 26 in the grid pattern 28 represents a separate DNA probe and constitutes a separate experiment. A plurality of such grid pattern comprises an array set 30. Reference or “target” DNA (or RNA) is spotted onto the glass slide 24 and chemically bonded to the surface. Fluorescently labeled “probe” DNA (or RNA) is introduced and allowed to hybridize with the target DNA. Excess probe DNA that does not bind is removed from the surface of the slide 24 in a subsequent washing process.
As illustrated in FIG. 2, a confocal laser microarray scanner or microarray reader is commonly used to scan the microarray slide 24 to produce one image for each dye used by sequentially scanning the microarray with a laser of a proper wavelength for the particular dye. Each dye has a know excitation spectra as illustrated in FIG. 3 and a known emission spectra as illustrated in FIG. 4. The scanner includes a beam splitter 32 which reflects a laser beam 34 towards an objective lens 36 which, in turn, focuses the beam at the surface of slide 24 to cause fluorescent spherical emission. A portion of the emission travels back through the lens 36 and the beam splitter 32. After traveling through the beam splitter 32, the fluorescence beam is reflected by a mirror 38, travels through an emission filter 40, a focusing detector lens 42 and a central pinhole 44. After traveling through the central pinhole 44, the fluorescence beam is detected by a detector, all in a conventional fashion.
The intent of a microarray experiment is to determine the concentrations of each DNA sample at each of the spot locations on the microarray. Further data analysis of the brightness values are typically done to produce a ratio of one dye's brightness to any or all of the other dyes on the microarray. An application of the microarray experiment is in gene expression experiments. Higher brightness values are a function of higher concentrations of DNA. With a microarray, a researcher can determine the amount a gene is expressed under different environmental conditions.
To be accurate, the reader must be able to quantitate the brightness of each microarray spot for each labeled DNA sample used in the experiment. To do this the reader must filter the emissions from any and all other fluorescent samples. The concentration of the DNA is a function of the brightness of the emission when excited by a laser of the proper wavelength. It becomes difficult to differentiate between the emissions of different dyes when the emission spectra of a dye overlaps with another. Furthermore, the brightness produced from the emission of one dye could be contaminated by emissions from another dye. This contamination of the brightness values is commonly known as crosstalk.
Microarray readers have been designed to simultaneously scan more than two dyes using lasers with the proper wavelength. In this type of experiment, multiple samples of DNA are hybridized onto the microarray, each with a different fluorescent label. Crosstalk contamination is equally likely as in the two dye experiments and can even be more troublesome when dyes with close emission spectra are placed on the same microarray.
U.S. Pat. Nos. 5,804,386 and 5,814,454 disclose sets of labeled energy transfer fluorescent primers and their use in multi-component analysis.
U.S. Pat. No. 5,821,993 discloses a method and system for automatically calibrating a color camera in a machine vision system.
The paper by Schena, M., et al., (1995) “Quantitative Monitoring of Gene Expression Patterns With a Complementary DNA Microarray”, Science 270; 467–469 is also related to the present invention.